CN1283238C - Use of alkannin in preparing medicine for treating tumor disease - Google Patents
Use of alkannin in preparing medicine for treating tumor disease Download PDFInfo
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Abstract
The present invention provides a new purpose of alkannin. The molecular weight of the alkannin is 288.2994; the chemical name is (+)-5, 8-dihydroxy-2-(1-hydroxy-4-methyl-3-pentenyl)-1, 4-naphthoquinone, and the molecular formula is C16H16O5. The alkannin provided by the present invention is combined with medical excipients or carriers, and the alkannin is applied to prepare the medicine for treating tumor diseases. The action of the alkannin on multi-medicine resistance tumor cells is free from the influence of p-glucoprotein; the killing effects of the alkannin on the multi-medicine resistance tumor cells are obviously superior to that of the existing common and representative anti-tumor medicines; the mechanism of the alkannin of killing the multi-medicine resistance tumor cells with Pgp high expression is that the cells are induced to be dead, and the mechanism of the cells is consistent with that of the parent medical sensitivity cells. The alkannin has low toxicity, and accordingly, the alkannin has the chemotherapy of the clinical tumors and has particularly favorable application prospects of the multi-medicine resistance of preventing the clinical tumors.
Description
Technical field
The purposes of pharmaceutically active ingredient in the invention belongs to relates generally to the new purposes of shikonin at the anti-multidrug resistance function of tumor medicine of preparation.
Background technology
Shikonin is one of main component of extracting from the root of Boraginaceae herbaceos perennial Radix Arnebiae (Radix Lithospermi), and Radix Arnebiae (Radix Lithospermi) all has distribution in the most of provinces and regions of China, and it has antiinflammatory, convergence and refrigeration function, medicinal its root, be applied to clinical, determined curative effect.Radix Arnebiae (Radix Lithospermi) is recorded in Shennong's Herbal at first, for preventing and treating the Chinese medicine of measles.Shikonin then is the main component of anti-inflammatory.Radix Arnebiae (Radix Lithospermi) is rich in shikonin and derivant thereof, and its content accounts for 3～6.5%, and color is dark red, and chemical constitution is the naphthoquinone class, can transmit meson in the redox reaction in vivo.Have hemostasis, antiinflammatory, antibiotic, antiviral and anticancer pharmacologically active.Multiple composition of Radix Arnebiae (Radix Lithospermi) and structures shape thereof the broad spectrum activity of its function and the variation of purposes, can become the intermediate feed of the natural series of products of multiple industry.
Shikonin, English shikonin by name, CAS number:517-89-5, molecular weight: 288.2994, chemical name is: (+)-5,8-dihydroxy-2-(1-hydroxy-4-methyl-3-pentenyl)-1,4-naphthoquinone, molecular formula is C
16H
16O
5, structural formula is as follows:
It has optical activity and iso-alkannin Alkannin is a pair of enantiomer.Shikonin is the acicular crystal that aubergine has metallic luster, is slightly soluble in water, is soluble in organic solvents such as benzene, ethanol, ether, and owing to the light that can absorb 500～560nm wavelength presents redness, and absorption spectrum can move with pH.It has ph stability, and solution colour changes along with pH, takes on a red color under the acid condition, and purple when neutral is blue during alkalescence, because colour stable, and LD own
50, be 3.0 ± 0.1g/kg to male mice, be 3.1 ± 0.1g/kg to female mice, reality is the avirulence component, is widely used in the pigment of food additive at present.And in as face breast, lip pomade cosmetics, also as natural pigment composition, this sees in the cosmetics of Japan more
[1,2]And just begin to utilize the cell engineering best cultivation to come the industrial-scale production shikonin as far back as Japan in 1984, as the raw material of cosmetics
[3]
For the medical usage of shikonin, go back the neither one systematic study, the pharmacological research of shikonin is mainly concentrated on antiinflammatory action, on the accelerated wound healing
[4～8]Though, find to have antitumor action very early for the research of active anticancer, the research of pharmacology is less, and Japanese scholar was arranged in 1977
[9]To the report of the anti-tumor activity of shikonin, but mechanism is also indeterminate.And the Chinese scholar was arranged in 1991
[10]Be used for the curative effect that clinical experiment treatment patient with advanced cancer has confirmed shikonin with the compound prescription of shikonin.The report of inducing the HL-60 apoptosis of tumor cells that shikonin was abroad just arranged to 1999
[11,12], and find to have in the apoptotic cell activation of the caspase Caspase 3 that plays a crucial role, and domesticly just have shikonin to induce the report of colorectal cancer cells apoptosis to calendar year 2001
[13]Do not see so far then that for the anti-multidrug resistance function of tumor of shikonin any report is arranged.
Multidrug resistance is meant that tumor cell produces cross tolerance to the medicine of the different structure and the mechanism of action, and it is the obstacle that becomes the chemotherapy success that tumor cell produces crossing drug resistant to multiple chemotherapeutics.According to incompletely statistics, the patient's cause of the death more than 90% is relevant with multidrug resistance (MDR).Wherein most typical mechanism is the high expressed of the protein called membrane transporters Pgp (P-glycoprotein) of ATP energy dependence.The high expressed of Pgp causes tumor cell that the different antineoplastic chemotherapy medicine of multiple 26S Proteasome Structure and Function such as purple triol, anthracene nucleus class, vinca etc. are developed immunity to drugs, its mechanism is that Pgp is the drug efflux pump on the cell membrane, to enter intracellular medicine and pump the extracellular, therefore cell obtains drug resistance.
Pgp separated a kind of molecular weight that obtains by Ling etc. in 1976 to be about the 170kDa glycosylated membrane protein from the Chinese hamster ovary cell of anti-Colchicine, because it can change the permeability of medicine, so called after P-glycoprotein (P is meant Permeability)
[14], be called for short Pgp.The Pgp overexpression of tumor cell is considered to most typical drug resistance mechanism up to now.
The transmembrane protein that Pgp is made up of 1280 amino residue, it is most typical abc transport albumen, has its general common structure.Obtained the Pgp structure of 25 dust resolution by ultramicroscope and single particle image analysis technology, data show Pgp be an intimate diameter be 10nm cylinder, Pgp by four obviously the zone form: two highly hydrophobic, and to stride the intracytoplasmic ATP binding of being positioned at of diaphragm area and two highly-hydrophilics regional.People's Pgp is by one of MDR gene family MDR1 coding, and this gene is positioned at chromosome 7q21.1.The MDR gene family comprises MDR1 and MDR3 (also claiming MDR2) in the mankind, Pgp is meant what MDR1 encoded, thereby Pgp is also referred to as MDR1.
In tumor cell, the overexpression of Pgp causes the minimizing of drug accumulation in the cell, thereby makes cell produce drug resistance.Pgp is a kind of medicine efflux pump, and by its ATP-binding site, produce power makes medicine discharge outside the born of the same parents against Concentraton gradient behind the hydrolysising ATP, therefore is that a kind of ATP relies on pump.
Pgp is protein that causes tumor multi-medicine drug-resistant the most completely of studying at present, important antineoplastic chemotherapy medicine clinically such as purple triol, amycin, epirubicin, epirubicin, vincristine, rice take off anthraquinone, rules and forms dimension (gleevec at present, STI571, imatinib), hydroxy camptothecin, Deng the substrate that is Pgp, drug effect all reduces greatly because of the expression of tumor Pgp.The expression of Pgp is the major reason of chemotherapy of tumors failure in the tumor cell.Therefore, the antitumor drug that the exploitation drug effect is not influenced by Pgp, the medicine of the multidrug resistance tumor of especially anti-Pgp high expressed is extremely important.We find that shikonin can kill the multidrug resistance tumor cells of Pgp high expressed effectively, the influence that its drug effect is not expressed by tumor cell Pgp, the drug effect that shikonin kills multidrug resistance cell significantly is better than purple triol, epirubicin, vincristine, mitomycin, clinical antitumor drug such as hydroxy camptothecin.
Summary of the invention
A kind of new purposes that the purpose of this invention is to provide shikonin, that is: the application of shikonin in preparation medicine for treating tumor thing.Shikonin that the present invention adopts extracts or chemosynthesis from plant such as Radix Arnebiae (Radix Lithospermi) according to prior art, and the medicine of preparation can contain other antitumor drug, and drug excipient or carrier.Shikonin of the present invention is the application in preparation antitumor cell multidrug resistance medicine mainly; The also application in preparation clinical tumor chemotherapeutics.
Described medicine can be made the dosage form of intestinal or non-intestinal combination medicine by this formulation art known method.Dosage form mainly comprises liquid preparation, granule, tablet, electuary, soft gelatin capsule, capsule, drop pill or injection.
The form of medication of preparation mainly comprises oral administration or drug administration by injection.Oral formulations comprises slow releasing agent.
When tumor cell produces multidrug resistance, high expressed Pgp in the cell, Pgp is a transmembrane protein, can intracellular antitumor drug such as purple triol, amycin, vincristine discharge extracellular will be entered, drug effect is reduced greatly, and shikonin is not subjected to the influence of p-glycoprotein to the effect of multidrug resistance tumor cells, and the lethal effect of multidrug resistance tumor cells obviously is better than purple triol, amycin, epirubicin, vincristine, hydroxy camptothecin etc.The mechanism that shikonin kills the multidrug resistance tumor cells of Pgp high expressed is cell death inducing, and is consistent with its parent's susceptibility cell.
The beneficial effect of the application of shikonin of the present invention in the preparation tumour medicine is as follows: (1) has potential applicability in clinical practice, and the drug effect that shikonin can kill and wound multidrug resistance tumor cells is significantly higher than existing clinical medicines such as purple triol, amycin, epirubicin, vincristine, hydroxy camptothecin; (2) shikonin has lower toxicity, and therefore, shikonin has the application prospect of good clinical chemotherapy of tumors, especially anti-clinical tumor multidrug resistance.
Description of drawings
Fig. 1 is amycin gathering in K562 and K562/Adr cell;
Fig. 2 is the Pgp expression of K562 and K562/ADR cell;
Fig. 3 is the growth of K562/ADR cell for shikonin suppresses K562;
Fig. 4 suppresses the cell growth of KB and KBv200 for shikonin;
Fig. 5 handles the dna ladder shape histogram of K562 and K562/Adr cell behind the cell, features of apoptosis for shikonin;
Fig. 6 is the metamorphosis after the shikonin element of 1 μ g/ml is handled K562 cell different time.Features of apoptotic form, karyopycnosis appear in cell;
Fig. 7 is the metamorphosis after the shikonin element of 1 μ g/ml is handled K562/Adr cell different time.Features of apoptotic form, karyopycnosis appear in cell;
Fig. 8 is that the shikonin of variable concentrations is handled flow cytometry analysis result behind K562 and the K562/Adr cell 72hr;
Fig. 9 is that the shikonin of variable concentrations is handled flow cytometry analysis result behind K562 and the K562/Adr cell 72hr;
Figure 10 is that the shikonin of 1 μ g/ml is handled Caspase3 behind the K562 cell 48hr, and 6,9 enzymatic activitys change;
Figure 11 is that the shikonin of 1 μ g/ml is handled Caspase3 behind the K562/Adr cell 48hr, and 6,9 enzymatic activitys change.
The specific embodiment
The invention will be further described below in conjunction with specific embodiment.
The drug resistance spectrum of embodiment 1 multidrug resistance cell K562/ADR cell strain is identified
Multidrug resistance cell strain K562/ADR system is set up by the inductive method of antitumor drug amycin in the erythroleukemia cell strain K562 of nineteen ninety-five personnel selection by people such as Hu flood
[15]
Mtt assay is measured the drug resistance situation of mdr cell to 11 kinds of typical antineoplastic agents
Get the good K562 that is in exponential phase of growth of growing way form and two kinds of cells of K562/ADR, counting, centrifugal, with 8 * 10
3The concentration in cell number/hole is inoculated in 96 orifice plates, it is 0.01 that every hole 100 μ l add final concentration respectively, 0.02,0.1,0.2,1, the amycin of 2,10,20 μ g/ml, epirubicin, daunorubicin, vincristine, paclitaxel, methotrexate, fluorouracil, ametycin, etoposide, hydroxy camptothecin, 11 kinds of representational antineoplastic agents such as homoharringtonine (every concentration is three parallel multiple holes of row all), every hole adds 100 μ l, cumulative volume 200 μ l, and establish blank and do not add medicine and only add the cell control wells are hatched jointly by after 72 hours, carry out mtt assay and detect the cell proliferation survival condition: every hole adds 2mg/ml MTT 58 μ l, cultivates 4hr; The centrifugal 10min of 1000r.p.m on the dull and stereotyped centrifuge, abandoning supernatant blots culture fluid, and every hole, back adds 200 μ l DMSO, bluish violet crystallization Formazan in the dissolved cell; After the vibration 5min crystallization dissolving, and survey the 570nm wavelength OD of place value on the microplate reader.
Reflect the survival cells amount with the OD value, calculate the survival rate of each cell behind each concentration drug effect
Survival rate=dosing hole OD value/empty OD value * 100% of no medicine contrast
Average, the correlation curve with Origin 7.0 softwares drafting drug level and cell survival rate obtains the half-inhibition concentration IC of various medicines to each cell
50, and calculate the drug resistance multiple (resistance factor) of mdr cell to various medicines with this
RF=IC
50 mdr cells/ I
The C50 sensitive cells
The result: K.562/ADR cell has multidrug resistance.To the anthracene ring antitumor medicinal daunorubicin, amycin and epirubicin and be the vincristine of targeting with the microtubule, paclitaxel, etoposide and homoharringtonine, mitomycin c all has higher drug resistance, and for the topoisomerase be the hydroxy camptothecin of targeting with the antimetabolite fluorouracil, methotrexate is drug resistance not basically then.The drug resistance spectrum the results are shown in Table 1.
The drug resistance spectrum of table 1 K562/ADR
| Medicine | K562IC 50 (μg/ml) | K562/ADR?IC 50 (μg/ml) | K562/ADR?RF |
| Daunorubicin | 0.030±0.011 | 0.189±0.064 | 6.3±0.2 |
| Amycin | 0.032±0.014 | 0.248±0.041 | 7.8±2.1 |
| Epirubicin | 0.026±0.006 | 2.549±0.016 | 98.0±22.0 |
| Etoposide | 2.840±0.083 | 15.069±0.022 | 5.3±0.2 |
| Fluorouracil | 1.515±0.0424 | 1.882±0.041 | 1.2±0.0 |
| Homoharringtonine | 0.016±0.07 | 0.116±0.003 | 7.3±3.0 |
| Hydroxy camptothecin | 0.103±0.058 | 0.298±0.036 | 2.9±1.3 |
| Methotrexate | ＜0.01±0.028 | ＜0.01±0.010 | |
| Ametycin | 0.033±0.019 | 0.395±0.085 | 12.0±4.3 |
| Paclitaxel | ＜0.01±0.008 | 0.413±0.054 | ＞41.3±27.6 |
| Vincristine | ＜0.01±0.004 | 0.603±0.210 | ＞60.3±3.12 |
Embodiment 2 flow cytometry analysis K562 and K562/ADR cell are to the ability of gathering of amycin
Get well-grown K562 and two kinds of cells of K562/ADR that are in exponential phase of growth, with final concentration be the amycin of 10 μ g/ml is cultivated 120min in incubator after, with cold PBS washing 2 times, collecting cell, counting is made into 1 * 10 with cold PBS
6The cell suspension of cells/ml, 4 ℃ of fluorescence intensities that are saved to amycin in the capable cells were tested by flow cytometry cell of sample, the detection excitation wavelength is 488nm, and accepting wavelength is 575nm, and the intensity arbitrary unit is represented doxorubicin concentration in the phase pair cell.
The ability that result: K562 gathers amycin is strong, behind the amycin co-cultivation 2hr of 10 μ g/ml, the interior doxorubicin concentration of K562 cell is about 5 times of K562/ADR cell, then because effluxing of Pgp causes drug resistance to produce, doxorubicin concentration is significantly less than sensitive cells to the K562/ADR cell in the cell.See Fig. 1.
Get well-grown K562 and two kinds of cells of K562/ADR that are in exponential phase of growth, with cold PBS washing 2 times, collecting cell, counting is made into 1 * 10 with cold PBS
6The cell suspension of cells/ml, add the monoclonal antibody that is combined with PE (phycoerythrin) labelling (R-PE-17F9 of BD company) of the anti-Pgp of 20 μ l respectively, lucifuge association reaction 15min under the room temperature adds PBS and washes twice, the capable flow cytometer of last sample is represented the expression of Pgp with fluorescence intensity.
The result: the K562 cell of negative control, the ratio of high expressed Pgp only has 0.68%, mdr cell K562/ADR then up to 88.79%.The expression of the average film albumen Pgp of while mdr cell K562/ADR is 302.40/9.65=31 a times of K562.Therefore the drug resistance mechanism of K562/ADR cell mainly is that the overexpression of Pgp causes, referring to Fig. 2.
Brief summary: the K562/ADR cell strain is to be the multidrug resistance cell strain of feature with the Pgp high expressed typically.
The comparison of the anti-multidrug resistance cell of medicines such as embodiment 4 shikonin and purple triol
Get the good K562 that is in exponential phase of growth of growing way form and two kinds of cells of K562/ADR, counting, centrifugal, with 8 * 10
3The cell concentration of cells/well is inoculated in 96 orifice plates, every hole 100 μ l.Adding final concentration respectively is 0.01,0.02,0.1,0.2,1,2,10, drug solutions such as the shikonin of 20 μ g/m, purple triol, amycin, vincristine, every hole adds 100 μ l, 3 parallel holes of every concentration row, putting into incubator hatched 72 hours, mtt assay detects, and the IC of shikonin to two kinds of cells tried to achieve in mapping
70Value.
The result: shikonin significantly is better than existing clinical medicines (table 2) such as purple triol, amycin, epirubicin, vincristine, hydroxy camptothecin to the drug effect of killing and wounding of multidrug resistance cell
Table 2. shikonin and other clinical chemotherapy medicines compare the drug effect of killing and wounding multidrug resistance tumor cells K562/Adr
| Medicine | IC 70(μg/ml) |
| Shikonin | 0.743±0.001 |
| Paclitaxel | 3.532±0.054 |
| Epirubicin | 9.256±0.062 |
| Amycin | 0.923±0.041 |
| Vincristine | 9.674±0.211 |
| Mitomycin | 1.866±0.085 |
| Hydroxy camptothecin | 16.641±0.036 |
| 5-fluorouracil | 5.623±0.037 |
Embodiment 5: shikonin is to the lethal effect of multidrug resistance cell and parent's susceptibility cell thereof
Multidrug resistance cell K562/Adr derives from susceptibility cell K562 cell, and both differences are the former high expressed p-glycoprotein, so the former has the multidrug resistance of pair tumor chemotherapeutic drug, and the latter does not have multidrug resistance; KBv200 and KB cell be another to multidrug resistance cell and susceptibility cell, the former high expressed p-glycoprotein.
Get growing way the form good K562 that is in exponential phase of growth, K562/ADR, KB and four kinds of cells of KBv, counting, centrifugal, with 8 * 10
3The cell concentration of cells/well is inoculated in 96 orifice plates, every hole 100 μ l.Adding final concentration respectively is 0.01,0.02,0.1,0.2, the shikonin solution of 1,2,10,20 μ g/m, and every hole adds 100 μ l, and 3 parallel holes of every concentration row are put into incubator and were hatched 72 hours, and mtt assay detects, and the IC of shikonin to two kinds of cells tried to achieve in mapping
50Value.
The result: from two amount effect relation curves that suppress growth, two curves overlap substantially, and IC
50Basic identical, K562 is 0.436 μ g/ml, K562/ADR is that 0.445 μ g/ml that is to say that shikonin is that the kill capability of K562/ADR does not have marked difference to sensitive cell line K562 and multidrug resistance, proves that MDR cell K562/ADR does not have toleration to shikonin.Shikonin is to the IC of two kinds of cells
50Be about 0.45 μ g/ml, when reaching 1 μ g/ml, two kinds of cells are killed off (see figure 3) basically.
Shikonin is suitable to the lethal effect of KB and KBv200, proves that also MDR cell KBv200 does not have the toleration (see figure 4) to shikonin.
These results show shikonin killing and wounding and suppress the influence that drug effect is not subjected to tumor multi-medicine drug-resistant tumor cell, especially be not subjected to the influence of tumor cell p-glycoprotein, even tumor cells expression p-glycoprotein, the drug effect of multiple antitumor drug is reduced greatly, still do not influence the antitumor drug effect of shikonin.
The mechanism of embodiment 6 shikonin artitumor multi-medicine-resistants-agarose gel electrophoresis detects genomic DNA
Get the good K562 that is in exponential phase of growth of growing way form and two kinds of cells of K562/ADR, counting, centrifugal, with every hole 1 * 10
6Cell number is inoculated in 6 orifice plates.Adding final concentration is the shikonin of 1 μ g/ml, and adding culture fluid, to complement to every hole cumulative volume be 6ml.Putting into incubator cultivates, and respectively at 0hr, 24hr, 48hr and 72hr collecting cell, wash twice with cold PBS, the centrifugal 5min of 3500r.p.m on the centrifuge, cell transfer to 1.5ml eppendorf pipe, is pressed test kit (Shen, Shanghai energy lottery industry Science and Technology Ltd.) and gone up explanation extraction genomic DNA.Get 10 μ l extracts, add 2 μ l 6 * DNA Loading Buffer, in 0.5ml eppendorf pipe behind the mixing on agarose gel (the 0.5g/50mlTAE buffer of sample to 1%, 2 μ l EB have been added), after running glue 90min under the 80V voltage, observed result and taking pictures under the ultraviolet imagery.
Result: induce K562 and drug-resistant cell strain K562/ADR after 24 hours with the shikonin of 1 μ g/ml, as seen two kinds of cells all have the dna degradation that causes owing to apoptosis is the distinctive Ladder phenomenon that small pieces forms, and over time, gradient is obvious more, small fragment is dense more, the mechanism that shikonin artitumor multi-medicine-resistant cell is described is identical with the mechanism of the quick tumor cell of drug resistance, promptly based on cell death inducing, referring to Fig. 5, wherein band 1:0 hour, band 2:24 hour, band 3:48 hour, band 4:72 hour.
Mechanism one shikonin of embodiment 7 shikonin artitumor multi-medicine-resistants is induced two kinds of apoptotic morphological observations
Collect the good K562 that is in exponential phase of growth and the two kinds of cells of K562/ADR of growing way form, counting, centrifugal, with every hole 1 * 10
6Cell number is inoculated in 6 orifice plates.Adding final concentration is 1 μ g/ml shikonin, and adding culture fluid, to complement to every hole cumulative volume be 6ml, respectively at 12 hours, and the cell 100 μ l in 24 hours and 48 hours collection one holes.Cell concentration is diluted to 4 * 10 with PBS
5About cells/ml, with pelleter the above-mentioned cell suspension that adds 100 μ l cell fixation on wave carrier piece, make print.Dyeing: with 1: 1 Wright and Giemsa complex staining liquid, add 10 times of poststaining 10～20min of phosphate buffer dilution of pH6.8, dry at the flowing water flushing slide back side.The cellular morphology that apoptotic process observed and took down by the oil mirror changes.
The result: induce K562 and mdr cell K562/ADR with the shikonin of 1 μ g/ml, compare with normal staining cell behind the 12hr, volume dwindles to some extent, and nuclear chromatin is all arranged towards nuclear membrane one side pyknosis, darkens, and this is that to be in apoptosis early stage.Visible nuclear further concentrates behind the 24hr, and chromosome breakage, forms the apoptotic body that several also do not leave cell, and this is the apoptosis medium cell.After the 48hr, the Secondary cases necrosis has taken place in visible cell, has cavity, cell that swelling distortion is arranged in the endochylema, even begins to break, and is in apoptosis late period this moment, sees Fig. 6 and Fig. 7, wherein A: contrast, B:12 hour, C:24 hour, D:48 hour.
The mechanism of embodiment 8 shikonin artitumor multi-medicine-resistants-apoptosis-related protein enzyme Caspase-3,6,9 active detections
Collection is in the cell counting of exponential phase, with every hole 2 * 10
6Cell number is inoculated in 6 orifice plates, wherein three holes in contrast, three hole dosings.Adding final concentration is the shikonin solution of 1 μ g/ml, adds culture fluid at last and complements to cumulative volume 6ml.Be cultured to be in transfer the phase cell of dying after, the piping and druming mixing, centrifugal collecting cell is operated according to detection kit (Caspase-3,6,9 color check test kits, BD company) description below.
Result: after the shikonin of 1 μ g/ml induces two kinds of cell 48hr most apoptotic cell to occur, detect Caspase enzymatic activity absorbance in the born of the same parents, can obviously find out the Caspase3 of two kinds of cells that are in the apoptosis phase, 6,9 enzyme all is activated, and the strongest with the activation degree of Caspase3.So shikonin is an inducing cell generation apoptosis, because as the core protein enzyme Caspase3 of apoptosis activation is arranged, the result is referring to Fig. 8,9.
Table 3 Caspase enzymatic activity testing result
Mechanism-flow cytometry analysis the apoptosis rate of embodiment 9 shikonin artitumor multi-medicine-resistants
Get the good K562 that is in exponential phase of growth of growing way form and two kinds of cells of K562/ADR, counting, centrifugal, with every hole 1 * 10
6Cell number is inoculated in 6 orifice plates.Add final concentration and be respectively 0.5,1, the shikonin of 5 μ g/ml, and adding culture fluid, to complement to every hole cumulative volume be 6ml, puts into incubator and cultivate, after cultivating 72hr, collecting cell washes twice with cold PBS, the centrifugal 5min of 2000r.p.m on the centrifuge, cell transfer to the streaming pipe, and is added the ethanol fixed cell of 1ml 70%.After 4 ℃ of refrigerator overnight, the centrifugal fixative that goes adds a little P BS, and the back adds several propidium iodides (PI), lucifuge reaction 10～20min, and the up flow type cell instrument detects apoptosis rate.
Result: during apoptosis, because the activation of Cobra venom endonuclease, cause chromatin extensively to rupture in the nucleosome junction, before cell is FCM with PI dyeing, the DNA of some degradeds is because the 70% alcoholic acid permeability that fixedly causes increases, and the DNA fragment is overflowed in cell, and the dna content of apoptotic cell reduces, dyeing back fluorescence intensity reduces, thereby the G on the DNA rectangular histogram
0/ G
1The apoptotic peak of a hypodiploid has appearred in phase the place ahead.For non-viable non-apoptotic cell then because the modal difference of swelling, can be on the Parameter Map of the reflection cellular morphology of FCM just in addition sorting.Referring to Figure 10, Figure 11, associative list 4 as can be known, two kinds of cells are after shikonin is induced 72hr, apoptosis all takes place, and apoptosis rate is the concentration dependence, from 60% of 11% to the 5 μ g/ml of 0.5 μ g/ml, and little with the shikonin of concentration to the apoptosis rate difference of two kinds of cells.A among Figure 10: contrast, B:0.5 μ g/ml shikonin, C:1 μ g/ml shikonin, D:5 μ g/ml shikonin; A:0.05 μ g/ml shikonin among Figure 11, B:0.5 μ g/ml shikonin, C:1 μ g/ml shikonin, D:5 μ g/ml shikonin.
The shikonin of table 4 variable concentrations is induced K562 and the apoptosis rate of K562/ADR cell after 72 hours
Above embodiment explanation: shikonin significantly is better than antitumor clinical medicines such as purple triol, epirubicin, vincristine, hydroxy camptothecin, mitomycin to the drug effect of multidrug resistance tumor cells; Shikonin kills the drug effect of multidrug resistance tumor cells and parental cell thereof does not have remarkable difference, the influence that the drug effect that proves shikonin is not expressed by tumor cell Pgp; After shikonin is handled Pgp high expressed multidrug resistance tumor cells, the genomic DNA agarose gel electrophoresis occur hypodiploid peak among Ladder feature, the flow cytometry analysis figure appearance, variation on the oily sem observation cellular morphology in dyeing back, and the activation of Caspase-3 enzyme, show that the mechanism of action that shikonin kills the multidrug resistance tumor is inducing cell generation apoptosis.These presentation of results shikonin have the medication preparation and the potential applicability in clinical practice of the multi-drug resistance of the tumor of wide anti-Pgp high expressed.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the various measuring methods of technical process, quality index and other similar change all belong to the scope of the invention.
The partial reference document that the present invention relates to:
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Claims (1)
1. the application of shikonin in preparation antitumor cell multidrug resistance medicine.
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| CN101194920B (en) * | 2006-09-29 | 2012-07-04 | 杭州贺博生物技术有限公司 | Lithospermum and application of its active ingredient in preparing medicament for treating tumour stem cell |
| CN101671376B (en) * | 2009-09-21 | 2013-04-17 | 浙江大学 | Alkannin acetyl glucose and preparation method and application thereof |
| CN102552296A (en) * | 2010-12-30 | 2012-07-11 | 浙江大学 | Application of alkannin glucoside to preparation of pyruvate kinase inhibitor |
| CN103284983A (en) * | 2013-05-10 | 2013-09-11 | 上海市肺科医院 | Application of alkannin and/or derivative thereof in preparing medicine for prohibiting cancer cell metastasis |
| CN112022841B (en) * | 2020-09-10 | 2022-03-11 | 吉林大学 | Iron/alkannin nano-composite, preparation method of supermolecule self-assembly of iron/alkannin nano-composite and application of iron/alkannin nano-composite |
| CN113181117B (en) * | 2021-03-22 | 2022-08-26 | 沈阳药科大学 | Shikonin and anthracycline chemotherapeutic drug co-carried liposome and preparation method and application thereof |
| CN113332265A (en) * | 2021-05-31 | 2021-09-03 | 大连医科大学 | IKK beta/NEMO small-molecule inhibitor and application thereof in preparation of drugs |
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